Reducing morbidity in surgical resections: Third-harmonic generation microscopy a

描述(申请人提供)：意外损伤神经是手术切除肿瘤和其他手术中发病率的主要原因之一。神经纤维在手术过程中很难与周围组织区分开来，这使得外科医生很难仔细地绕过它们，避免它们受到伤害。广义地说，这项拟议工作的目标是建立三次谐波产生(THG)显微镜，作为术中高灵敏度显示神经纤维的一种方法。我们之前已经证明，THG提供了一种高灵敏度的方法来对啮齿动物中枢神经系统中的髓鞘进行成像，并已将其用于在体脊髓中的髓鞘成像。我们最近收集的初步数据表明，这种方法也可以突出周围神经。最终，这种成像方式可以让外科医生可视化神经纤维，然后仔细地在它们附近工作，从而防止与神经损伤相关的手术并发症。我们确定了完成这项工作所必需的三个具体目标。首先，我们在初步数据的基础上，确定了用THG成像显示有髓周围神经束的最佳激光和成像参数集。其次，我们通过比较使用和不使用这种新的成像方式的啮齿动物的前列腺切除手术的结果来评估THG成像对保留神经的有效性。在这个目标中，我们进一步建立在以前的结果的基础上，不仅检查神经在THG可视化时有多好，而且在用双光子激发荧光(2PEF)显微镜成像时肿瘤是如何完全提取的。至关重要的是，THG成像所需的激光和成像系统与2PEF成像完全兼容，允许这两种模式协同工作，帮助外科医生最大限度地切除肿瘤并将神经损伤降至最低。综合起来，这两个目标将确立我们的神经成像方法的实用性，但在手术中使用 房间环境显然需要开发一种比我们目前的显微镜更不笨重的仪器。为此，在第三个目标中，我们设计了一种内窥镜，该内窥镜针对THG(以及2PEF)成像进行了优化，并可用于人类的腹腔镜手术(如许多前列腺癌切除)。综上所述，这项工作将确立THG作为一种可行的外科成像手段的实用性，并将开发出可用于手术室的第一代仪器。如果成功，这项工作可以极大地减少切除手术的术后发病率--特别是在关键神经靠近切除部位的情况下，例如在前列腺--从而改善患者的预后。

DESCRIPTION (provided by applicant): Unintentional injury to nerves is one of the primary causes of morbidity during surgical resection of tumors as well as in other surgeries. Nerve fibers are difficult to distinguish from the surrounding tissues during surgery, making it difficul for surgeons to carefully work around and avoid their injury. Broadly speaking, the goal of the proposed work is to establish third-harmonic generation (THG) microscopy as an approach to visualize, with high sensitivity, nerve fibers intraoperatively. We have previously shown that THG provides a high-sensitivity approach to imaging myelin in the central nervous system of rodents and have used this for in vivo imaging of myelin in the spinal cord. We recently collected preliminary data that shows this approach can highlight peripheral nerves as well. Ultimately, this imaging modality could allow the surgeon to visualize nerve fibers and then work carefully near them, thereby preventing surgical complications associated with nerve injury. We identify three Specific Aims necessary to complete this work. First, we build on our preliminary data and identify the best set of laser and imaging parameters to visualize myelinated peripheral nerve bundles with THG imaging. Second, we assess the effectiveness of THG imaging to spare nerves by comparing the outcome of prostate resection surgeries in rodents done with and without this novel imaging modality. In this Aim, we further build on previous results and examine not just how well nerves are spared when visualized with THG, but also how completely tumors are extracted when they are imaged with two-photon excited fluorescence (2PEF) microscopy. Critically, the laser and imaging systems necessary for THG imaging are completely compatible with 2PEF imaging, allowing these two modalities to work in concert to help a surgeon both maximize tumor extraction and minimize nerve damage. Taken together, these two aims will establish the utility of our approach to nerve imaging, but use in an operating room environment will clearly require development of a less-unwieldy instrument than our current microscopes. Toward this end, in the third Aim, we design an endoscope that is optimized for THG (as well as 2PEF) imaging and could be used in laparoscopic surgeries (such as many prostate cancer resections) in humans. Taken together, this work will establish the utility of THG as a viable imaging modality for use in surgery and will develop the first-generation instrument that could be used in an operating room. If successful, this work could dramatically reduce the post-surgical morbidity of resection surgeries - especially in cases where critical nerves are close to the resection site, such as in the prostate - and therefore improve patient outcomes.